Ex-Th17 (Nonclassical Th1) Cells Are Functionally Distinct from Classical Th1 and Th17 Cells and Are Not Constrained by Regulatory T Cells

Sharee A. Basdeo, Deborah Cluxton, Jamal Sulaimani, Barry Moran, Mary Canavan, Carl Orr, Douglas J. Veale, Ursula Fearon and Jean M. Fletcher
J Immunol March 15, 2017, 198 (6) 2249-2259; DOI: https://doi.org/10.4049/jimmunol.1600737

Abstract

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly resistant to suppression of proliferation and cytokines by regulatory T cells. Finally, we showed that ex-Th17 cells accumulated in the joints of rheumatoid arthritis patients. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the rheumatoid arthritis joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Introduction

There is accumulating evidence in the literature implicating the dysregulation of Th17 cells in the pathogenesis of many chronic inflammatory conditions, such as rheumatoid arthritis (RA), inflammatory bowel disease, and psoriasis (1, 2). As a result, there has been intense interest in therapeutic strategies designed to block Th17 cells. Effective targeting of Th17 cells requires a full understanding of how these cells behave at sites of inflammation. It has emerged that Th17 cells are relatively unstable and can exhibit functional plasticity, particularly at sites of autoimmune inflammation. Th17 cells can transition through a stage of producing both their signature cytokine IL-17 in addition to IFN-γ, and then lose expression of IL-17 to resemble a Th1-like cell that produces IFN-γ in the absence of IL-17 (3, 4). Th17 lineage cells have been shown to develop from a CD161+ precursor and retain its expression after switching to what has been called an ex-Th17 or nonclassical Th1 phenotype (5, 6). The common origin of Th17 and ex-Th17 cells has also been demonstrated at the epigenetic level (7). A consensus in the literature suggests that bona fide Th17 cells can be sorted based on their coexpression of CD161, CCR6, and CCR4 in the absence of CXCR3 expression (8, 9). Th1 cells can be sorted by CXCR3 and CCR5 expression in the absence of CD161, CCR6, and CCR4 (8, 9). Plastic Th17 cells that have switched into producing IFN-γ maintain the expression of CD161 and CCR6 and gain the expression of CXCR3 (8, 9). Only one study to date has investigated the differences between bona fide Th1 cells and plastic ex-Th17 or nonclassical Th1 cells in terms of their expression of IFN-γ and IL-17, transcription factors and chemokine receptors (10). This article shows that nonclassical Th1 cells can be distinguished from bona fide Th1 cells using a combination of surface markers; however, functional differences between the classical and nonclassical Th1-type cells have not yet been fully investigated.

Ex-Th17 cells constitute the majority of tissue-infiltrating CD161+ CD4+ T cells in the joints of autoimmune arthritis patients (11, 12) and affect the gut of Crohn’s disease sufferers (13, 14) and the cerebrospinal fluid of MS patients (15). These studies suggest that Th17 cell plasticity may occur locally; however, because ex-Th17 cells also exist in the peripheral blood, trafficking of these cells from the periphery into inflamed tissues cannot be ruled out. The role of these ex-Th17/nonclassical Th1 cells in human autoimmunity is not yet clear, and no data have been published to date analyzing the pathogenicity of bona fide Th1 cells compared with plastic ex-Th17 or nonclassical Th1 cells.

We have previously shown that CD161+ Th17 lineage cells, which include bona fide Th17 cells, Th17 cells transitioning to ex-Th17 cells (t-Th17 cells), and fully switched ex-Th17 cells, accumulate in the inflamed synovium of RA patients (16). In healthy controls (HCs), CD161+ Th17 lineage cells exhibited a number of pathogenic features including increased longevity, polyfunctional cytokine production, and resistance to regulatory T cell (Treg)–mediated suppression (16). In this study, we have further dissected the CD161+ Th17 lineage population into bona fide Th17 cells and ex-Th17 cells to compare their features and susceptibility to Treg-mediated suppression with classical Th1 cells. We show that ex-Th17 cells are in fact functionally distinct from Th1 cells and exhibit features that are pathogenic in autoimmunity. Ex-Th17 cells retain some features of their Th17 precursors such as their differentiated memory phenotype and increased capacity for survival. However, the previously identified brake on Th17 cell proliferation (17) was lifted upon the switch to the ex-Th17 phenotype. Furthermore, we show in this study that the previously identified resistance to Treg-mediated suppression of total CD161+ Th17 lineage cells (16) is accounted for by a resistance of ex-Th17 rather than bona fide Th17 cells, because both the latter and Th1 cells were effectively suppressed by Tregs. Finally, we show that the enrichment of CD161+ Th cells in the RA joint was accounted for by ex-Th17 cells, rather than bona fide Th17 cells, which were found at similar frequencies as in peripheral blood. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the RA joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Materials and Methods

Th1, Th17, and ex-Th17 cell sorting

Leukocyte-enriched buffy coats from anonymous healthy donors were obtained with permission from the Irish Blood Transfusion Board, St. James’s Hospital, Dublin, Ireland. CD4+CD45RO+ memory T cells were purified from PBMC using MACS (Miltenyi Biotec). CD4+CD45RO+ cells were stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, and CCR6 (eBioscience) to sort Th1, Th17, and ex-Th17 subpopulations. CD4+CD45RO+CD25−CD161−CXCR3+CCR4−CCR6− Th1, CD4+CD45RO+CD25−CD161+CXCR3−CCR4+CCR6+ Th17, and CD4+CD45RO+CD25−CD161+CXCR3+CCR6+ ex-Th17 cells were sorted from HC PBMCs on a FACSAria Fusion (BD Biosciences) cell sorter, with purities routinely >95% (see Supplemental Fig. 1).

Flow cytometry

Lymphocytes were identified by forward and side scatter, and dead cells and doublets were excluded. Cells were stained extracellularly with amine-binding markers for dead cells (fixable viability dye; eBioscience) and fluorochrome-conjugated Abs specific for CD4, CD8, CCR7, CD161, CD25 (eBioscience), CD3, CCR6 (BD Biosciences), CD45RO (Miltenyi Biotec), CXCR3, and CCR4 (BioLegend). For Ki67 and Bcl2 (eBioscience) analysis, cells were surface stained ex vivo, then fixed and permeabilized for intranuclear staining (FOXP3 staining buffer kit; eBioscience). For intracellular cytokine analysis, cells were stimulated for 5 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 μg/ml; Sigma-Aldrich). Cells were then surface stained, fixed, and permeabilized using Caltag Fix and Perm kit (BD Biosciences) before staining intracellularly for cytokines GM-CSF (BioLegend), IFN-γ, IL-2 (BD Biosciences), IL-17 (eBioscience and BD Biosciences), and TNF (eBioscience). The CD4+ T cell population in PMA/ionomycin-stimulated samples was gated on the basis of CD3+CD8− cells because CD4 was downregulated upon stimulation. Cells were acquired on a BD FACSCanto II or LSRFortessa (BD Biosciences) and analyzed with FlowJo software with gating confirmed on unstimulated control samples or fluorescence minus one controls, as appropriate. Presentation of polyfunctional cytokine production was performed using SPICE version 5.3 software (National Institute of Allergy and Infectious Diseases, National Institutes of Health) (18).

ELISA

Th1, Th17, and ex-Th17 cells (sorted as described earlier) were stimulated with anti-CD3 (1 μg/ml) and allogeneic irradiated (60 Gy) PBMCs to serve as APCs. Cells were maintained in humidified incubators at 37°C with 5% CO2 for 5 d. Supernatants were harvested and the concentrations of IFN-γ, IL-17, GM-CSF, and TNF were quantified by ELISA (Ready-Set-Go kits; all eBioscience), according to the manufacturer’s protocol.

T cell cloning

Th1, Th17, and ex-Th17 cells were sorted (as described earlier) into 96-well plates with one cell per well. Cells were also cultured at 100 and 0 cells per well to provide comparisons for growth of cell clones. Cells were stimulated with anti-CD3 (1 μg/ml) and irradiated (60 Gy) PBMCs (2 × 105 cells per well). All wells were treated with IL-2 (20 IU/ml) every 2–3 d, and the medium was changed as required.

Suppression assays

CD4+ T cells or CD4+CD45RO+ memory T cells were purified from buffy coat–derived PBMCs using MACS (Miltenyi Biotec). CD4+ T cells were stained with fluorochrome-conjugated Abs specific for CD39, CD25, CD4, and CD127 (eBioscience) to sort Tregs. CD4+CD25+CD127loCD39+ Tregs were routinely >90% FOXP3+. CD4+CD45RO+ cells were stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, and CCR6 (eBioscience) to sort Th1, Th17, and ex-Th17 subpopulations, as described earlier. Sorted Th1, Th17, and ex-Th17 cells were labeled with CellTrace Violet (CTV; Life Technologies), according to the manufacturer’s protocol, and cultured at 5 × 104 cells per well alone or in coculture with equal numbers of unlabeled autologous Tregs in the presence of anti-CD3 (1 μg/ml; eBioscience) and 3 × 105 CFSE-labeled irradiated (60 Gy) allogeneic PBMCs to serve as APCs.

Cells were maintained in humidified incubators at 37°C with 5% CO2 for 4–5 d. Proliferation of responder cells was measured by CTV dilution. Cells were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 μg/ml; Sigma-Aldrich) to assess cytokine production. CFSE-labeled irradiated APCs and unlabeled Tregs were excluded from the analysis. Percent suppression of proliferation (as measured by CTV dilution) was calculated as the difference in proliferation of responder cells cultured alone and cocultured with Tregs, over the frequency of proliferating responder cells cultured alone: Responder − (responder+Treg)/(responder) × 100. Percent suppression of cytokine production was assessed in a similar manner from proliferating cells only.

RA patients

All patients fulfilled the American College of Rheumatology 2010 (19) criteria for a diagnosis of RA and had active disease at time of sampling (as classified by disease activity in 28 joints [DAS28] scores and/or erythrocyte sedimentation rate and C-reactive protein). Fully informed, written consent was obtained from each patient. This study was approved by the St. Vincent’s University Hospital Ethics and Medical Research Committee. PBMCs and synovial fluid mononucleocytes (SFMCs) were isolated by density gradient centrifugation (Lymphoprep; Axis-Shield poC). The ratio of female to male patients in the cohort was 3:2 with a median age of 59 y (range 19–83 y). These patients had a disease activity score (DAS28) of 4.2 ± 0.26 with 22.4% naive to treatment, 65.3% on synthetic disease-modifying antirheumatic drugs (DMARDs; e.g., methotrexate and sulfasalazine), and 40.1% on biologic DMARDs. No significant difference in DAS28 was observed between patients on no treatment compared with those treated with DMARDs or biologics.

Statistical analysis

Statistical analyses were performed using Prism 5 software. Two groups within a sample were determined by Student paired t test with two-tailed p values; three or more groups were analyzed by one-way ANOVA with Tukey’s multiple comparison test. Statistical differences between two groups, each containing more than one variable, were determined by two-way ANOVA with Bonferroni posttests. The p values <0.05 were considered significant.

Results

Sorting of Th1, Th17, and ex-Th17 cells on the basis of CD161 and chemokine receptor expression

To distinguish and sort Th1, Th17, and ex-Th17 cell populations, we used their differential expression of CD161 and the chemokine receptors CXCR3, CCR6, and CCR4 (9, 10). Memory CD4 T cells were sorted and stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, and CCR6. CD4+CD45RO+CD25−CD161−CXCR3+CCR4−CCR6− Th1, CD4+CD45RO+CD25−CD161+CXCR3−CCR4+CCR6+ Th17, and CD4+CD45RO+CD25−CD161+CXCR3+CCR6+ ex-Th17 cells were cell sorted according to the gating strategy shown in Fig. 1A and Supplemental Fig. 1. The sorted cells were stimulated with PMA/ionomycin and brefeldin A to determine their production of IL-17 and IFN-γ (Fig. 1B). Sorted Th1 cells produced IFN-γ and no IL-17; Th17 cells produced IL-17 and virtually no IFN-γ, whereas ex-Th17 cells produced IFN-γ to a similar extent as Th1 cells and a small amount of IL-17 (Fig. 1B). The ex-Th17 cell population likely included transitioning ex-Th17 cells, which are in an intermediate state between Th17 and ex-Th17 cells, and therefore still produced IL-17 or IL-17 plus IFN-γ simultaneously. In addition, sorted Th1, Th17, and ex-Th17 cells produced very little IL-4 or IL-10, as shown by intracellular cytokine staining and ELISA (Supplemental Fig. 2). To further characterize the cytokine profile of Th cell subsets, we stimulated cells with anti-CD3 plus irradiated APC for 5 d, and cytokines in the supernatant were measured by ELISA (Fig. 1C). The cytokine profiles of Th1 and Th17 cells were in accordance with what would be expected for these subsets. Th1 cells produced the most IFN-γ and virtually no IL-17 (Fig. 1C). Th17 cells produced significantly more IL-17 than Th1 cells (p < 0.001) and also some IFN-γ, most likely reflecting the plasticity of the Th17 cells during the culture period. Ex-Th17 cells, which would include both transitioning IL-17+IFN-γ+ cells as well as fully switched IL-17−IFN-γ+ cells, produced both IFN-γ and IL-17 (Fig. 1C). Ex-Th17 cells produced more GM-CSF and TNF (p < 0.001) than either Th17 or Th1 cells. These data validated the sorting protocol for Th1, Th17, and ex-Th17 cell populations and suggested that despite their reduction in IL-17 production, ex-Th17 cells may produce higher amounts of other proinflammatory cytokines.

FIGURE 1.
FIGURE 1.

Th1, Th17, and ex-Th17 subpopulations isolated on the basis of the expression of cell surface markers produce the appropriate signature cytokines. PBMCs were isolated from healthy donors. Cells were enriched for CD4+ CD45RO+ memory Th cells using MACS and stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, and CCR6. CD4+CD45RO+CD25−CD161−CXCR3+CCR4−CCR6− Th1, CD4+CD45RO+CD25−CD161+CXCR3−CCR4+CCR6+ Th17, and CD4+CD45RO+CD25−CD161+CXCR3+CCR6+ ex-Th17 cells were sorted according to the gating strategy shown in (A). Sorted cells were stimulated with PMA/ionomycin in the presence of brefeldin A, stained intracellularly with fluorochrome-conjugated Abs specific for IL-17 and IFN-γ, and analyzed by flow cytometry (B). Cells were stimulated with anti-CD3 and irradiated APCs for 5 d. The concentrations of cytokine in the supernatant were measured by ELISA (C). *p < 0.05, **p < 0.01, ***p < 0.001.

Th17 cells switch into ex-Th17 cells in vitro

Having validated the strategy for sorting Th cell subsets, we next investigated the plasticity of Th17 cells in vitro. Th1 and Th17 cells were sorted as shown in Fig. 1A. Cells were stimulated with anti-CD3/irradiated APCs and exogenous IL-2 for 18 d. The concentrations of IL-17 and IFN-γ were assessed by ELISA on days 5, 12, and 18. Th1 cells produced similarly high levels of IFN-γ throughout, and IL-17 was undetectable (Fig. 2A). Sorted Th17 cells produced similar levels of IL-17 throughout; however, their production of IFN-γ increased significantly with time in culture (days 5–12, p < 0.001; days 12–18, p < 0.05). Cytokine profiles were also analyzed by flow cytometry after restimulation with PMA/ionomycin on days 0, 7, and 14. Sorted Th1 cells produced high levels of IFN-γ and no IL-17 (Fig. 2B). On day 0, sorted Th17 cells produced IL-17 and a low frequency produced IFN-γ; however, by days 7 and 14, there was an increase in the frequencies of IL-17+IFN-γ+ cells and IFN-γ+IL-17− cells (Fig. 2B). These data suggest that a proportion of sorted IL-17+IFN-γ− Th17 cells switch during in vitro culture to become IL-17+IFN-γ+ and IL-17−IFN-γ+ cells. Because a low frequency of cells appeared to be producing IFN-γ on day 0, it cannot be concluded that the population of IL-17−IFN-γ+ arose solely from bona fide Th17 cells because the small population observed on day 0 may have expanded by day 7. Therefore, to definitively prove that Th17 cells are plastic in vitro, Th17 cells were single cell cloned, expanded in culture, and then restimulated for analysis of IL-17 and IFN-γ production at weeks 4 and 8. By week 4, the majority of cells produced IL-17 alone, whereas some produced both cytokines or IFN-γ alone (Fig. 2C). By week 8, however, very few cells produced IL-17 and the majority of cells (74%) produced IFN-γ only (Fig. 2C). This reduction in IL-17 in parallel with increasing IFN-γ production was confirmed by ELISA (Fig. 2C). Taken together, these data confirm that in the absence of Th17-skewing cytokines, Th17 cells undergo plasticity during in vitro culture, losing their expression of IL-17 and gaining expression of IFN-γ.

FIGURE 2.
FIGURE 2.

Th17 cells switch into ex-Th17 cells in vitro. PBMCs from HC donors were sorted into Th1 and Th17 cells according to the gating strategy shown in Fig. 1. Cells were cultured for 18 d in the presence of anti-CD3, irradiated APCs, and IL-2. Cells were restimulated with fresh anti-CD3 and irradiated APCs on days 7 and 14. The concentrations of IFN-γ and IL-17 present in the supernatants were measured by ELISA on days 5, 12, and 18. *p < 0.05, ***p < 0.001 (A). Cells were stimulated with PMA/ionomycin, stained intracellularly with fluorochrome-conjugated Abs specific for IL-17 and IFN-γ, and analyzed by flow cytometry on days 0, 7, and 14 postsorting (B). Th17 cells were sorted (one cell per well) into 96-well plates (C). Single-cell clones were stimulated with anti-CD3 and irradiated APCs, fed with IL-2, and allowed to grow for 8 wk. Cells were stimulated with PMA/ionomycin, stained intracellularly with fluorochrome-conjugated Abs specific for IL-17 and IFN-γ, and analyzed by flow cytometry on weeks 4 and 8 postsorting. Representative dot plots show plasticity of a Th17 clone in vitro (C). The concentrations of IFN-γ and IL-17 present in the supernatant on weeks 2 and 4 were determined by ELISA (C, right).

Phenotypic and functional features of ex-Th17 cells

Th17 cells have previously been described as having stem cell–like features despite having an effector memory phenotype (20, 21); however, it is not clear whether these features are retained upon the switch to the ex-Th17 phenotype. We therefore analyzed the memory phenotype of Th1, Th17, and ex-Th17 cells. PBMCs from healthy donors were stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, CCR6, CCR7, Bcl2, and Ki67 and analyzed by flow cytometry. Th1, Th17, and ex-Th17 subpopulations were gated as described in Fig. 1A.

A significantly higher frequency of Th17 (p < 0.01) and ex-Th17 (p < 0.05) cells had an effector memory phenotype (CD45RO+CCR7−) compared with Th1 cells (Fig. 3A). Both Th17 (p < 0.01) and ex-Th17 (p < 0.001) cells had significantly higher expression of antiapoptotic Bcl-2 compared with Th1 cells (Fig. 3B). These data indicated that similar to Th17 cells, ex-Th17 cells display a differentiated effector memory phenotype and retain the increased capacity for survival of Th17 cells.

FIGURE 3.
FIGURE 3.

Ex-Th17-type cells are phenotypically distinct from classical Th1 cells and proliferate more than bona fide Th17 cells. PBMCs from healthy donors were stained with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD25, CD161, CXCR3, CCR4, CCR6, CCR7, Bcl2, and Ki67 and analyzed by flow cytometry. Th1, Th17, and ex-Th17 subpopulations were gated as described in Fig. 1. (A) The percentage of memory cells with an effector memory phenotype (CCR7−) in each subpopulation (n = 7). (B) Expression of Bcl2 (indicated as median fluorescence intensity [MFI]) in subpopulations (n = 10). (C) Frequency of Ki67 expression in subpopulations (n = 21). (D) Th1, Th17, and ex-Th17 subpopulations were sorted, labeled with CTV, and cultured for 4 d with anti-CD3 and irradiated APCs. The frequency of proliferating cells was measured by CTV dilution. The graph shows the percentage of proliferating cells in the subpopulations (n = 4), and the representative histograms (E) show the dilution of CTV. *p < 0.05, **p < 0.01, ***p < 0.001.

Th17 cells have previously been shown to have reduced proliferation compared with Th1 cells, because of their increased expression of IL-4I1 (17). We therefore compared the proliferation of the cell subsets both ex vivo and after stimulation of sorted subsets in vitro. The frequency of cells cycling ex vivo was analyzed by staining for Ki67 and flow cytometry. Significantly less Th17 cells were in cycle compared with Th1 cells (p < 0.001), whereas there was no difference between Ki-67 expression in ex-Th17 cells compared with either Th1 or Th17 cells (Fig. 3C). Sorted cell subsets were also stained with CTV and stimulated with anti-CD3 plus irradiated APCs, and the frequency of proliferating cells was measured by CTV dilution on day 3. Ex-Th17 cells proliferated significantly more than Th17 cells (p < 0.05) (Fig. 3D, representative histograms in Fig. 3E). These data suggest that the previously described brake on Th17 cell proliferation may be lifted when they switch to become ex-Th17 cells. Thus, ex-Th17 cells retain some features of their Th17 cell precursors including a differentiated memory phenotype and stem cell–like characteristics; however, they acquire increased proliferative capacity.

Ex-Th17 cells display significantly more cytokine polyfunctionality compared with Th1 and Th17 cells

We have previously shown that CD161+ Th17 lineage cells, which include both Th17 and ex-Th17 cells, were more polyfunctional for cytokine production than CD161− Th cells (16). In addition, the data from Fig. 1C suggested that ex-Th17 cells produced increased amounts of GM-CSF and TNF compared with Th1 or Th17 cells. Thus, we next analyzed the production of multiple proinflammatory cytokines by Th1, Th17, and ex-Th17 cells. Th1, Th17, and ex-Th17 subpopulations were sorted and cultured for 5 d with anti-CD3 plus irradiated APCs. Cells were stained with fluorochrome-conjugated Abs specific for GM-CSF, IFN-γ, IL-2, IL-17, and TNF, analyzed by flow cytometry, and results were collated using SPICE software (Fig. 4). The pie charts represent the average frequencies of active cytokine-producing cells producing every possible combination of the five cytokines analyzed (Fig. 4A). The segments within the pie chart denote populations producing different combinations of cytokines and are heat map coded (Fig. 4A, gray-red in pie segment legend) to indicate increasing polyfunctional cytokine production. The size of the pie segment relates to the frequency of the particular population. The arcs around the circumference indicate the particular cytokines (see arc legend) produced by the proportion of cells that lie under the arc (Fig. 4A). Parts of the pie surrounded by multiple arcs represent polyfunctional cells. The frequency of cells producing the combination of IFN-γ, TNF, GM-CSF, and IL-2 was significantly increased in ex-Th17 cells relative to Th1 (p < 0.05) and Th17 (p < 0.001) cells, as was the frequency of cells producing the combination of IFN-γ, GM-CSF, and TNF. These data indicate that although ex-Th17 cells have lost the expression of IL-17, they are more polyfunctional than either Th1 cells or their Th17 cell precursors.

FIGURE 4.
FIGURE 4.

Ex-Th17 cells display significantly more cytokine polyfunctionality compared with Th1 and Th17 cells. Th1, Th17, and ex-Th17 subpopulations were sorted and cultured for 5 d with anti-CD3 and irradiated APCs. Cells were stained with fluorochrome-conjugated Abs specific for GM-CSF, IFN-γ, IL-2, IL-17, and TNF, analyzed by flow cytometry, and results were collated using SPICE software. (A) The pie charts represent the average frequencies of active cytokine-producing cells producing every possible combination of the five cytokines analyzed. The segments within the pie chart denote populations producing different combinations of cytokines and are heat map coded (pie legend: gray-red) to indicate increasing polyfunctional cytokine production. The size of the pie segment correlates to the frequency of the particular population. The arcs around the circumference indicate the particular cytokine (see arc legend) produced by the proportion of cells that lie under the arc. Parts of the pie surrounded by multiple arcs represent polyfunctional cells. (B) The abridged bar graph shows frequencies (>1%) of combinations of cytokines produced by Th1 (white bars), Th17 (gray bars), and ex-Th17 (black bars) cells. The colors in the bar below correspond to the pie colors in (A) and indicate the number of cytokines produced. *p < 0.05, **p < 0.01, ***p < 0.001.

Ex-Th17 cells are resistant to suppression by Tregs

Having previously shown that CD161+ Th17 lineage cells as a whole were resistant to Treg-mediated suppression (16), we next compared the susceptibility of ex-Th17, Th17, and Th1 cells to suppression by Tregs. CD4+CD45RO+ T cells from HCs were sorted into Th1, Th17, and ex-Th17 Treg-depleted responder subsets that were labeled with the tracer dye CTV, cocultured in the presence or absence of sorted CD4+CD127loCD25+CD39+ Tregs, and stimulated with anti-CD3 and irradiated APCs. Representative histograms show that CTV dilution (proliferation) within Th1, Th17, and ex-Th17 populations cultured alone was similar on day 5 (Fig. 5A). Whereas coculture of Tregs inhibited proliferation of Th1 and Th17 responder cells, proliferation of ex-Th17 responder cells was relatively unaffected (Fig. 5A). Treg suppression of cytokine production was also examined after restimulation, and representative plots of intracellular cytokine staining are shown in Fig. 5B. Although Treg suppression of TNF production by Th1 and Th17 cells was observed, there was no suppression of TNF produced by ex-Th17 responder cells (Fig. 5B). Collated data (n = 11) show significantly reduced Treg-mediated suppression of proliferation of ex-Th17 cells compared with Th1 (p < 0.01) and Th17 (p < 0.05) cells (Fig. 5C). Collated data (n = 8–11) demonstrated relative resistance to Treg-mediated suppression of TNF (p < 0.01), GM-CSF (p < 0.05), IFN-γ (p < 0.05), and IL-17 (p < 0.05) production by ex-Th17 responder cells (Fig. 5D). These data indicate that compared with Th1 and Th17 cell populations, ex-Th17 cells were significantly more resistant to Treg-mediated suppression of proliferation and cytokine production.

FIGURE 5.
FIGURE 5.

Ex-Th17 cell proliferation and proinflammatory cytokine production are resistant to Treg-mediated suppression. Th1, Th17, ex-Th17, and CD4+CD25+CD127lo CD39+ cells (Tregs) were sorted. Responder cells were labeled with CTV and cultured alone or cocultured with Tregs in the presence of anti-CD3 and irradiated APCs. Cells were analyzed by flow cytometry; CTV+ cells were gated and proliferation was quantified by CTV dilution. (A) Representative histograms showing the frequency of cell proliferation and the percent of Treg-mediated suppression in parentheses. (B) Cells were restimulated, stained for intracellular cytokine production, and analyzed by flow cytometry; CTVlo cytokine+ cells were gated. Representative dot plots showing the frequency of TNF produced by the proliferating cell population and the percent suppression in parentheses. (C) Percent Treg-mediated suppression of Th1, Th17, and ex-Th17 cell proliferation (n = 11). (D) Percent Treg-mediated suppression of cytokines produced by proliferating Th1, Th17, and ex-Th17 cells; TNF (n = 8), GM-CSF (n = 9), IFN-γ (n = 9), and IL-17 (n = 11). *p < 0.05, **p < 0.01.

Accumulation of ex-Th17 cells in the joints of patients with RA

Having demonstrated that ex-Th17 cells exhibited features that would be considered pathogenic in the context of autoimmunity, we next investigated their frequency at the site of inflammation in patients with RA. Mononuclear cells from blood (PBMCs) and SF (SFMCs) were isolated from RA patients with active disease and compared with PBMCs from HCs. Cells were stained ex vivo with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD161, CXCR3, CCR4, and CCR6 and analyzed by flow cytometry. To assess cytokine production, we stimulated cells with PMA and ionomycin in the presence of brefeldin A, stained them with fluorochrome-conjugated Abs specific for CD3, CD8, CD161, IFN-γ, and IL-17 and analyzed them by flow cytometry. The frequency of CD161+ CD4 T cells was significantly increased in SFMCs compared with matched PBMCs from RA patients (Fig. 6A) (p < 0.001). In addition, the frequencies of Th1, Th17, and ex-Th17 cells were determined by the expression of cell surface markers, as described earlier, in untreated RA patients (n = 2; Fig. 6B). Ex-Th17 cells (CD161+ CXCR3+ CCR6+) were significantly increased in SFMCs compared with matched PBMCs, whereas Th1 and Th17 cells were not significantly different in the circulation compared with the site of inflammation (Fig. 6B). The expression of CD161 in combination with IFN-γ or IL-17 was used to identify the frequencies of Th1, Th17, and ex-Th17 cells in the total patient cohort rather than the expression of chemokine receptors, which were perturbed in many of the activated synovial cells, and thus could not be reliably used to identify T cell subsets. Th1 cells were identified as CD161−IFN-γ+, Th17 cells as CD161+IL-17+IFN-γ−, and ex-Th17 cells were CD161+IFN-γ+IL-17−, whereas t-Th17 cells were CD161+IFN-γ+IL-17+ (representative dot plots in Fig. 6B, 6C). There were no significant differences in the frequencies of Th1, Th17, t-Th17, and ex-Th17 cells between PBMCs from HCs versus RA patients (Fig. 6D). In addition, there was no significant difference in the frequency of Th17 cells within SFMCs versus PBMCs from HCs or RA patients. There was, however, a significant increase in ex-Th17 cells with SFMCs compared with PBMCs from RA patients (p < 0.01) or controls (p < 0.05) (Fig. 6D) and a trend toward an increase in t-Th17 cells (Fig. 6D). In addition, Th1 cells were significantly increased in SFMCs compared with PBMCs from both RA patients and HCs (p < 0.05) (Fig. 6D).

FIGURE 6.
FIGURE 6.

Ex-Th17, but not bona fide Th17, cells are enriched in the joints of patients with RA. PBMCs and SFMCs were isolated from RA patients with active disease and compared with PBMCs from HCs. Cells were stained ex vivo with fluorochrome-conjugated Abs specific for CD4, CD45RO, CD161, CXCR3, CCR4, and CCR6 and analyzed by flow cytometry. To assess cytokine production, we stimulated cells with PMA and ionomycin in the presence of brefeldin A, stained them with fluorochrome-conjugated Abs specific for CD3, CD8, CD161, IFN-γ, and IL-17, and analyzed them by flow cytometry. (A) Dot plots showing the expression of CD161 in PBMCs and SFMCs from RA patients. Graph shows the frequencies of CD4+ cells expressing CD161 in PBMCs and matched SFMCs from RA patients (n = 5). (B) Graph shows the frequencies of Th1 (CD161− CXCR3+ CCR4− CCR6−), Th17 (CD161+ CXCR3− CCR4+ CCR6+), and ex-Th17 (CD161+ CXCR3+ CCR6+) cells in the peripheral blood and matched SF of untreated RA patients (n = 2). (C) Dot plots showing the production of IFN-γ (top) or IL-17 (bottom) versus CD161 in total CD4+ cells. (D) Representative plots showing the frequency of CD161+ cells expressing IL-17 versus IFN-γ (top panel) and CD161− cells expressing IL-17 versus IFN-γ (bottom panel). (E) Graphs showing the frequency of CD4+ CD161− cells expressing IFN-γ (classical Th1) in HC PBMCs (n = 13), RA PBMCs (n = 6), and RA SFMCs (n = 13); the frequency of CD4+ CD161+ expressing IL-17+ (bona fide Th17) in HC PBMCs (n = 16), RA PBMCs (n = 8), and RA SFMCs (n = 12); the frequency of CD4+ CD161+ coexpressing IL-17 and IFN-γ (transitioning ex-Th17) in HC PBMCs (n = 10), RA PBMCs (n = 7), and RA SFMCs (n = 11); and the frequency of CD4+ CD161+ expressing IFN-γ+ (nonclassical Th1/ex-Th17) in HC PBMCs (n = 16), RA PBMCs (n = 8), and RA SFMCs (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001.

These data showing a lack of enrichment of Th17 cells in the RA joint in parallel with an accumulation of both t-Th17 and ex-Th17 cells suggest that Th17 cells within the RA joint are highly plastic and are driven toward an ex-Th17 phenotype. Furthermore, as described earlier, ex-Th17 cells exhibit a number of pathogenic characteristics, hence their accumulation in the RA joint is likely to contribute to disease pathogenesis.

Discussion

In this study, we investigated the consequences of Th17 cell plasticity in the context of autoimmunity. We demonstrated that ex-Th17 cells retain the previously described stem cell–like features of Th17 cells, but also gain additional pathogenic characteristics including increased proliferative capacity, cytokine polyfunctionality, and resistance to Treg suppression. We also showed that these ex-Th17 cells accumulated within the inflamed joints of RA patients, where they seem likely to contribute to disease pathogenesis. Thus, despite sometimes being called nonclassical Th1 cells, these ex-Th17 cells are functionally distinct from both Th1 cells and their Th17 cell precursors.

Th1, Th17, and ex-Th17 cells can be identified either on the basis of CD161 and their expression of IL-17 or IFN-γ, or on the basis of CD161 and their expression of chemokine receptors CXCR3, CCR6, and CCR4 (9, 10). The former cannot be used to sort viable cells; therefore, for functional experiments, we sorted according to the latter strategy. In contrast, the expression of chemokine receptors can be perturbed in activated cells; therefore, we used CD161 and cytokine expression to identify the Th cell subsets from the inflamed RA joint. The sorting strategy for Th1, Th17, and ex-Th17 cells was validated by determining the cytokine profiles of sorted cells. As expected, after stimulation, Th1 cells produced IFN-γ, but not IL-17. Sorted Th17 cells produced their signature cytokine IL-17, in addition to some IFN-γ. We have observed that Th17 cells very quickly undergo plasticity in vitro, and Th17 clones were observed to switch from an IL-17+IFN-γ− to a mainly IFN-γ+IL-17− phenotype. Thus, it is likely that during the culture period sorted Th17 cells would have begun to switch phenotype. Sorted ex-Th17 cells, which would have included both transitioning IL-17+IFN-γ+–producing cells as well as fully switched IFN-γ-only–producing cells, produced both of these cytokines. Interestingly, however, ex-Th17 cells appeared to produce GM-CSF and TNF in higher amounts than either Th17 or Th1 cells. Given the well-established pathogenic roles of TNF and GM-CSF in autoimmunity, these data indicate that ex-Th17 cells may be key producers of these cytokines at sites of inflammation.

In agreement with previous studies (20), we showed that Th17 cells exhibited a more differentiated phenotype than Th1 cells, and this was shared with ex-Th17 cells. Given their terminally differentiated phenotype, it might be assumed that Th17 cells have a short half-life and display an exhausted or senescent phenotype, but, to the contrary, human Th17 cells have been shown to express high levels of stem cell–associated genes including Bcl2, HIF-1α, Notch, and Nanog (20). These data indicate that Th17-type cells are long-lived with a high capacity for self-renewal and resistance to apoptosis, characteristics that are likely to enhance their pathogenicity in the context of autoimmunity. We investigated whether these stem cell–like features were retained after switching to the ex-Th17 phenotype. Similar to Th17 cells, we found that ex-Th17 cells had significantly increased expression of antiapoptotic Bcl-2 compared with Th1 cells, suggesting an enhanced capacity for survival, despite their differentiated phenotype. The expansion of Th17 cells has been shown to be limited by their expression of IL-4I1, which provides a proliferative brake (17). Consistent with this, we confirmed that ex vivo Th17 cells expressed lower levels of the cell cycle molecule Ki67 compared with Th1 cells. However, cell tracer experiments revealed that ex-Th17 cells proliferated to a greater extent than Th17 cells, suggesting that the switch to the ex-Th17 phenotype may be accompanied by a lifting of the brake on proliferation. These data indicated that ex-Th17 cells shared some of the features of their precursors, including a differentiated phenotype, yet enhanced capacity for survival, while gaining an increased proliferative capacity.

We had previously shown that total CD161+ Th17 lineage cells were highly polyfunctional in terms of their cytokine production (16). In this study, we showed that despite losing the majority of their IL-17 production, ex-Th17 cells were more active in producing cytokines and were more polyfunctional for cytokine production than either Th1 or Th17 cells. These data indicate that ex-Th17 cells are highly proinflammatory.

Previous data indicated that total CD161+ Th17 lineage cells (which contain both bona fide Th17 cells and ex-Th17 cells) from HCs were relatively resistant to suppression by Tregs (16). Furthermore, we had demonstrated reduced suppression of effector cells from the RA joint, which could be restored upon depletion of CD161+ effector cells. In this study, we sought to dissect this further by comparing the susceptibility of Th1, Th17, and ex-Th17 cell subsets with Treg-mediated suppression. Interestingly, ex-Th17 cells were significantly more resistant to Treg-mediated suppression of proliferation and cytokine production, whereas no differences were observed between Th1 and Th17 cells. These data indicated that our previously observed CD161+ Th cell resistance to Treg suppression was accounted for by resistance of ex-Th17 rather than bona fide Th17 cells. This is also in agreement with an earlier study showing suppression of IL-17 by CD39+ Tregs (22). In addition to the resistance to Treg suppression shown in this article, plastic Th17 cells expressing the multidrug resistance marker/P-glycoprotein have also previously been shown to exhibit resistance to glucocorticoid suppression (23). This further supports the concept that ex-Th17 cells exhibit a variety of features likely to contribute to their pathogenicity in autoimmunity. The mechanism for resistance to Treg-mediated suppression observed in this study and our previous study (16) is still unknown, although we excluded a number of possibilities including a role for soluble factors, the CD161 molecule, and death or conversion of Tregs. We did, however, show that resistance to suppression was dependent on STAT3, because it could be reversed with a STAT3 inhibitor (16). Interestingly, drug resistance in cancer cells has also been shown to be driven by STAT3, raising the interesting possibility that resistance to Tregs and steroids in ex-Th17 cells may be mediated via a common mechanism. In support of a role for STAT3 in mediating resistance to Treg suppression in ex-Th17 cells, STAT3 activation in effector T cells via IL-6 signaling has previously been shown to mediate resistance to Treg suppression (24). However, because we could not demonstrate a role for soluble factors such as IL-6 in mediating the Treg resistance of ex-Th17 cells, the STAT3 activation may be mediated via an alternative mechanism or be an inherent feature of these cells.

Th17 plasticity has been recognized as a feature of autoimmune inflammation in juvenile arthritis, Crohn’s disease, and MS (11–14, 15), whereas we have previously shown a significant enrichment of total CD161+ Th cells within inflamed RA joints (16). We showed in this study in our cohort of RA patients that this enrichment of CD161+ Th cells was accounted for by an increase in ex-Th17 rather than bona fide Th17 cells. To our knowledge, no other studies have specifically analyzed ex-Th17 cells in RA SF; however, our finding that CD4+IL-17+ Th17 cells were not increased in RA SF compared with blood are consistent with two previous studies (25, 26). Yamada et al. (25) showed an increase in CD4+IFN-γ+IL-17−, but not CD4+IFN-γ−IL-17+, cells in SF compared with blood from RA patients. It is possible that the enriched CD4+IFN-γ+IL-17− population observed in the latter study included ex-Th17 in addition to Th1 cells. However, it is important to note that other studies have shown an increase in Th17 cells in SF from RA patients (27, 28). These relative differences in the frequencies of Th subsets in the RA joint could be reflective of differences in disease duration and activity, where Th17 cells may be increased early in disease and then increasingly switch to an ex-Th17 phenotype later in disease. Our cohort of patients was relatively advanced in disease stage, which could account for the higher frequency of ex-Th17 versus Th17 cells observed. In contrast, other studies that have shown Th17 cells to be enriched in the RA joint may have used patients at an earlier disease stage (27, 28). Although the majority of our patient cohort was undergoing therapy, none was on treatments that would specifically target Th17 cells. Furthermore, we also examined matched PBMCs and SFMCs from two untreated RA patients, and data supported the finding that ex-Th17 cells, but not bona fide Th17 cells, were enriched in SFMCs relative to PBMCs. Therefore, it is unlikely that the frequency of Th17 versus ex-Th17 cells was skewed as a result of therapy.

In addition, it would be of interest to determine whether biologic therapies will target pathogenic ex-Th17 cells in RA. It has been shown that anti-TNF induces IL-10 expression in CD4 T cells including Th17 cells (29). Because we have shown in this study that ex-Th17 cells produce high levels of TNF, determining the effect of anti-TNF therapy on ex-Th17 cells may give more insight into the mechanisms regulating these cells in vivo. Furthermore, GM-CSF is emerging as an important target in RA, with anti–GM-CSF trials currently in progress (30). Our data indicate that ex-Th17 cells rather than Th17 or Th1 cells are the main source of GM-CSF, and thus it is likely that anti–GM-CSF may constrain ex-Th17 cells. In contrast, anti–IL-17 would not be expected to inhibit ex-Th17 cells that no longer produce significant amounts of IL-17. Future studies examining the relative frequencies of Th cell subsets in the inflamed RA joint may allow for patients to be stratified for treatment with the most appropriate biologic therapy based on their expression of key cytokines.

Taken together, our data show that ex-Th17 cells exhibit pathogenic characteristics including enhanced survival, proliferation, and cytokine production, and importantly are not constrained by Tregs. Given that these cells accumulate at sites of autoimmune inflammation, it suggests that they exert a pathogenic role in autoimmunity. Furthermore, these findings have implications for therapeutic inhibition of Th17 cells, because anti–IL-17 would not target pathogenic ex-Th17 cells. Indeed, Th17 plasticity is a key feature in both RA and inflammatory bowel disease (13, 14, 16), where anti–IL-17 therapy has not proven to be particularly effective (31, 32). Thus, strategies to target Th17 lineage cells rather than IL-17 may be more effective in inhibiting the pathogenic effects of both Th17 and ex-Th17 cells in autoimmune diseases such as RA.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by Science Foundation Ireland Starting Investigator Grant BI593 (to J.M.F.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CTV
    CellTrace Violet
    DAS28
    disease activity in 28 joints
    DMARD
    disease-modifying anti-rheumatic drug
    HC
    healthy control
    RA
    rheumatoid arthritis
    SF
    synovial fluid
    SFMC
    synovial fluid mononucleocyte
    Treg
    regulatory T cell
    t-Th17 cell
    Th17 cell transitioning to ex-Th17 cell.

  • Received April 25, 2016.
  • Accepted January 9, 2017.

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